PROJECT SUMMARY Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are now widely employed to dis- cover the mechanisms of heart diseases and identify potential drug targets. A challenge for current iPSC- CM applications, however, is how to identify and promote functionally mature myocytes that can more faith- fully recapitulate human adult cardiomyocyte characteristics. To propel the next stage of discoveries, there is a critical need for methods that can derive mature iPSC-CM that can accurately model adult heart dis- ease phenotypes, but current efforts are hampered by a dearth of molecular markers that can serve as sur- rogate readouts of iPSC-CM functional maturity. Accordingly, the goal of the present F32 fellowship proposal is identify protein markers that can re- flect the status of in vitro functional maturation of human iPSC-CMs. iPSC-CMs gradually acquire functional- ly mature characteristics following prolonged periods in culture. We recently discovered 190 membrane- protein-encoding genes that are significantly induced at the transcript level in iPSC-CMs after prolonged (30-90 days) of culturing in vitro. Here I will test the hypothesize that a subset of these prolonged culture signatures (PCS) represent bona fide maturity markers of human cardiomyocytes and thus may be har- nessed to isolate functionally mature iPSC-CMs. To achieve this goal, I propose two specific aims: In Aim 1 I will employ high-resolution mass spectrometry to determine genes which are enriched in culture and in adult hearts at the protein-level, and which can potentially distinguish and isolate functionally mature sub- populations. In Aim 2 I will verify protein expression of the candidate markers at the single-cell level, and further evaluate the functional characteristics of iPSC-CMs isolated using protein markers, to compare the functional maturity and homogeneity of the acquired iPSC-CMs against current standards. The anticipated payoff of the proposed experiments will be an improved molecular understanding of iPSC-CM functional maturity in culture, which may lead to methods to isolate more mature iPSC-CM popu- lations that can be used for disease modeling studies. These goals are significant in my opinion because they have the potential to greatly improve current iPSC-CMs applications and open doors to development of engineering approaches to further enhance iPSC-CM production. At the same time, the proposed research training plan will also provide valuable training opportunities in stem cell biology (with Sponsor Dr. Joseph Wu) and single-cell analysis (with Co-Sponsor Dr. Garry Nolan), which will complement my existing exper- tise in proteomics and aid me in my future goal of setting up an independent research group in cardiovascu- lar medicine.